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Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland
Published on: December 1, 2015
Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland
Luigi Strizzi1, Mario Mancino, Caterina Bianco
1Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, Bethesda, Maryland 20892, USA.
Abstract:
Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.
Insights
Netrin-1 promotes mammary gland development and functional differentiation. It influences Cripto-1 and Nanog expression in multipotent cells, suggesting a role in mammary epithelial cell differentiation.
Area of Science:
- Developmental Biology
- Cell Biology
- Stem Cell Biology
Background:
- Netrin-1 regulates Cripto-1 (Cr-1) function and mammary gland development.
- Cr-1 is a target gene of Nanog and Oct4, key developmental regulators.
Purpose of the Study:
- Investigate the relationship between Netrin-1, Cr-1, Nanog, and Oct4 during mouse mammary gland development.
- Determine Netrin-1's role in mammary epithelial cell differentiation.
Main Methods:
- Histological analysis of transgenic and wild-type mice at different developmental stages.
- Immunostaining and Western blot analysis for protein expression.
- In vitro studies using HC-11 mammary epithelial cells and mouse embryonic stem (ES) cells.
Main Results:
- Exogenous Netrin-1 induced alveolar-like structures and enhanced alveologenesis.
- Netrin-1, Nanog, and Oct4 were expressed in embryonic mammary epithelium, while Cr-1 was in the mesenchyme.
- Netrin-1 expression was highest during lactation, inversely correlating with Cr-1 and Nanog levels.
- Netrin-1 stimulation increased beta-casein expression in mammary epithelial cells.
- Netrin-1 treatment reduced Nanog and Cr-1 levels and increased beta-III tubulin in differentiating ES cells.
Conclusions:
- Netrin-1 plays a role in mammary gland development and functional differentiation.
- Netrin-1 may influence Nanog and/or Cr-1 expression in multipotent mammary gland cells.
- Netrin-1 facilitates functional differentiation of mammary epithelial cells.
